Essential oils and morphological study of Mentha aquatica

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1 Pharmacognosy Communications Volume 4 Issue 2 Apr Jun Research Article Essential oils and morphological study of Mentha aquatica Adel Nadjib Chaker, Habiba Boukhebti, Rachid Sahraoui and Messaoud Ramdhani Laboratoire de Valorisation des Ressources Biologiques Naturelles (VRBN), Département Biologie et Ecologie Végétale, Faculté des sciences de la nature et de la vie UFA Sétif Algérie ABSTRACT: As part of the development of plant resources, we are interested in the chemical study of essential oils of Mentha aquatica that is widely used in Algerian traditional medicine. Essential oil extracted from this plant was obtained by hydrodistillation with a Clevenger type apparatus for a period of three hours. The extraction produced yellowish essential oils with a very strong odor. Chemical analysis of the essential oils was achieved by gas chromatography coupled with mass spectrometry (GC/MS). The major compound present in the essential oil of M. aquatica was found to be linalyl acetate (26.1 %), other constituents included α-pinene (22.7 %), Linalol (13.8 %). The morphological study of M. aquatica shows two different types of glandular trichomes on both leaves and stems but lacunar parenchyma is only present in stem section. KEYWORDS: Essential oils, Chemical analysis, M. aquatica, (water mint), Morphological study. INTRODUCTION Most of mentha species are perennial herbs growing wildly in damp and wet places throughout temperate regions of Eurasia, North Africa, Australia and South Africa. [1] The plants belong to Mentha genus is considered as an industrial group as they are a source of essential oils. They are rich in certain monoterpenes, which are widely used in food as flavour, cosmetic and pharmaceutical industries. Several mentha species are used across the globe for their medicinal and culinary properties. They usually taken after a meal for their ability to reduce indigestion and colonic spasms. [2] In addition to countering colds, coughs, constipation, stomach-ache or inflammation of the appendix, stomach trouble and heart complaints. [3] Mentha aquatica (Water Mint) is a perennial plant of genus Mentha. It is herbaceous rhizomatous plant growing to 90 cm tall. The stems are square in cross sections, green or *Correspondence ID: chakeran@yahoo.fr DOI: /pc purple, and variably hairy to almost hairless. The rhizomes are widely spread fleshy and bear fibrous roots. The leaves are ovate to ovate-lanceolate, 2 6 cm long and 1 4 cm wide, green (sometimes purplish), opposite, toothed and vary from hairy to nearly hairless. Flowers are tiny, densely crowded, purple, tubular, and pinkish to lilac in color. Flowering period starts in mid to late summer. The plant is pollinated by insects and also spreads by underground rhizomes, as with other species of mint. All parts of the plant have a distinctly minty smell. [4] The aim of this study is to identify the chemical composition of essential oil and the anatomical study (glands trichomes) of M. aquatica that is widely used in Algerian traditional medicine, M. aquatica is used as tea to treat cough, digestive diseases and cold. MATERIALS AND METHODS Plant material The aerial parts of Mentha aquatica at flowering stage were collected from north east of Algeria. These were washed with tap water, to eliminate soil and other surface contaminants and then air-dried at shade in ambient temperature. The plant material was subsequently cut into small pieces for extraction. Extraction of the essential oil The aerial parts of the plant were subjected to hydrodistillation for 3 h using a Clevenger-type apparatus. The oil 34 Copyright 2014 EManuscript Publishing Services, India

2 obtained was collected and dried over anhydrous sodium sulfate and stored at 4 C in sealed brown vials until analysis. The oil analysis was carried out using GC-FID and GC/MS. Gas chromatography (GC-FID) analysis The gas chromatography analysis of the two volatile oils was performed using a HP 6890 equipped with: Flame ionization detectors (FID), HP Innowax (DB5; polyethylenglycol) 30 m 9 x 0.25 mm ID, 0.25 μm film thickness fused capillary column. The carrier gas was Hydrogen: (1ml min -1). The oven temperature program was 5 min isothermal at 50 C, then C (BP- 20) at rate of 5 C/min and held isothermally (300 C) for 5 min. The injection port temperature was 280 C, detector 300 C. Volume injected: 1 μl of 1: 60 (v: v in hexane) solution. Percentages of the constituents were calculated by electronic integration of FID peak areas. Gas chromatography-mass spectrometry (GC/MS) analysis The analyses of the volatile constituents were run on a Hewlett-Packard GC-MS system (GC: 7890; MSD-HP 5975-C.). The fused-silica DB5 capillary column (30 m 9 x 0.25 mm ID, film thickness of 0.25 μm) was directly coupled to the MS. The carrier gas was helium, with a flow rate of 1.0 ml min-1. Oven temperature was programmed (50 C for 5 min, then C at 5 C/min) and subsequently, held isothermally (300 C) for 5 min. Injector port: 250 C, detector: 280 C, split ratio 1:100. Volume injected: 1 μl of 1 % solution (diluted in hexane): HP 5975 recording at 70 ev; scan time 1.5 s; mass range amu. The identification of the oil components was based on comparison of their mass spectra with those of NIST mass spectral library, [5,6] and those described by, [7] as well as on comparison of their retention indices either with those of authentic compounds or with literature values. [7]. Preparation of Sections for Anatomical Study The anatomical study was realized on young fresh parts of stems and leaves and sections were taken manually using sambucus wood, sharp riser blade and double coloration. [8] RESULTS AND DISCUSSION Chemical composition The analysis of the essential oils of M. aquatica by GC and GC/MS identified 43 components corresponding to 97.8 % of the total oil (Table 1), the gas chromatogram of the oil on a HP-5 MS capillary column is shown in (Figure 1). The major compound in essential oil of M. aquatica is linalyl acetate (26.1 %), followed by α-pinene (22.7 %), linalol (13.755%), α-terpeneol (3.42%) and geranyl butyrate (3.39%). In the Table 1: chemical composition for Mentha aquatica essential oil. Pic RT % Compounds α-thujene α- Pinene Fenchene Camphene Sabinene β- Pinene Myrcene Delta-3-Carene α-terpinene Para-cymene Limonene Eucalyptol Z- β- Ocimene E- β- Ocimene Gamma Terpinene Terpinolene Linalol α-terpinolene Terpineol Terpinene-4-ol α-terpeneol Trans-geraniol Pulegone Acetate de linalyle Acetate de lavandulyle Geranyl octanoate Geranyl butyrate Mentha-1,4,8-triene Elemene<BETA-> β- Caryophyllene Trans-b -Farnesene α- Humulene Germacrene-D β- Cadinene Elemol Gamma-cadinene Epi-globulolemophilene α-selinene Eudesmol Abietatriene Sandra copimarinal Zierene Ferruginol Total

The reason for these variations in essential oils composition may be attributed to factors related to ecotype, the environment including temperature, relative humidity, irradiance, photoperiod, [17]

Typically, the predominant essential oil pattern consists of acyclic monoterpenes like linalool, 2-oxygenated monoterpenes like carvone, and 3-oxygentated monoterpenes for example menthol.

9%) were recognized as the main component whereas the major compounds of the leaf oil of the this plant were piperitenone oxide (25.7%), β-caryophyllene (12.0%) and 1.8-cineole (10.3%).

3 The reason for these variations in essential oils composition may be attributed to factors related to ecotype, the environment including temperature, relative humidity, irradiance, photoperiod, [17] the period of collection and the part of plant used. [11] Tuker et al., 2007 [18] said that the genetics of the essential oil pathways and the enzymes involved are well known in mentha. Typically, the predominant essential oil pattern consists of acyclic monoterpenes like linalool, 2-oxygenated monoterpenes like carvone, and 3-oxygentated monoterpenes for example menthol. Figure 1: Chromatogram of M. aquatica essential oil obtained by GC stem essential oil of M. aquatica from Iran, β-caryophyllene (22.4%), viridiflorol (11.3%) and 1, 8-cineole (10.9%) were recognized as the main component whereas the major compounds of the leaf oil of the this plant were piperitenone oxide (25.7%), β-caryophyllene (12.0%) and 1.8-cineole (10.3%). [9] The composition of essential oil of in the present study is different from those reported in recent [2, 10] studies. The identical Botanical plant in fact can give more or less important different extracts, biochemical specificity and proprieties of some plants are varied in different regions. [11] In present study, results are compared with other previous studies and examined the similarities and the differences of this plant in different regions and with other species that belong to the same genus. Znini M. et al, 2011 [12] found in their study that in M. spicata essential oils from Morocco carvone (29 %) and transcarveol (14 %) were the main components. Another study by Derwich et al, 2012 [13] showed that piperitone (35.6 %) and menthol (40.5 %) were the major constituents of Mentha pulegium and Mentha rotundifolia essential oils respectively. Gulluce et al. [1] reported cis-piperitone epoxide (18.4 %), followed by pulegone (15.5 %) and piperitenone oxide (14.7 %) as the main compounds of essential oil from M. longifolia, growing in Turkey. A further study [14] showed that piperitone (38 %) and piperitenone (33 %) were the main compounds of Mentha pulegium essential oil; in addition, results published by Vian et al. [15] present that pulegone (83.70 %) was the main compound of M. pulegium essential oil. Similar result was found by Zaks et al. [16] whereas the main compounds of M. aquatica var, citrata volatiles are the monoterpenes (alcohol linalool and its ester linalyl), as well as the results of Gulluce et al. [1] The essential oils of mint genus mainly consisted of oxygenated monoterpenes such as carvone, menthone were the principal oxygenated monoterpenes in the essential oils of M. spicata, M pulegium. Morphology of the Plant Morphological variations were observed among the plants growing in the natural habitat. Plant height varies from 45 to 80 cm and grows in moist damp streams. The plant is a perennial and aromatic with squared stems and green opposite short-stalked leaves. Flowering period is between August and October. Anatomical Study The anatomical study which was performed on young fresh stems showed the presence of pith, vascular stacks, cortex and epidermis whereas collenchyma tissue (supporting tissue) is existed in the cortex in four corners of the stem (Figure 3); The cortex is characterized by lacunous parenchyma tissue. However, in leaves, vascular bundles are surrounded by lacunous parenchyma tissue (Figure 6). Observations by light microscope show the epidermis layer have stoma joined with subsidiary cells to form stomatal complex. Both stems and leaves of this plant contain glandular and covering trichomes (Figures 3 7). Two types of trichomes at different developmental stages: simple hairs (covering trichomes) and secretive glands with short stalks are present (Figure 7,8). The first is unicellular and very small with globule head (capitate, glandular trichomes); the second is big and multi-cellular (peltate glandular trichomes). Peltate glandular trichomes Figure 2: M. aquatic general view. 36

Figure 3: Cross sections of stem of M. aquatica (A) general view, (B) lacunous parenchyma tissue in cortex.

aquatica (pel) peltate glandular trichome, (cap) capitate glandular trichome, (Sto) stomata. Figure 4: Cross sections of stem of M.

Figure 8: glandular and covering trichomes on young leaves of M. aquatic A: covering trichomes, B: glandular trichomes.

Simple hairs are composed of elongated cells with a large base cell.

adaxial side in young and mature leaves. Figure 5: Cross sections of M.

In aged leaves and stems, the density of the three types of glandular trichomes were lower compared to young shots and varies with the

4 Figure 3: Cross sections of stem of M. aquatica (A) general view, (B) lacunous parenchyma tissue in cortex. Figure 7: glandular trichomes on leaves of M. aquatica (pel) peltate glandular trichome, (cap) capitate glandular trichome, (Sto) stomata. Figure 4: Cross sections of stem of M. aquatica (A), capitates and peltate glandular trichomes, (B), (C) and (D) peltate glandular trichomes. Figure 8: glandular and covering trichomes on young leaves of M. aquatic A: covering trichomes, B: glandular trichomes. are bigger than capitate trichomes. Covering trichomes are simple and multi-cellular. Simple hairs are composed of elongated cells with a large base cell. At mature stage, the peltate glandular trichome is composed of a disc of eleven cells surmounted by a large sub cuticular storage space (Figure 3B,D). peltates glandular trichomes were more abundant on abaxial surface of the leaf while capitates glandular trichomes were more dense on adaxial side in young and mature leaves. Figure 5: Cross sections of M. aquatica stems (glandular and covering trichomes), A: covering trichomes, B: glandular trichomes. In aged leaves and stems, the density of the three types of glandular trichomes were lower compared to young shots and varies with the side of the leaves. Hatamneia et al. [19] reported that stomata on both epidermises of intercostal regions and leaves are amphistomatic (adaxial and abaxial surface), except for Ballota nigra subsp curdica, Mentha aquatica and Mentha longifolia, however, they are absent at the veins of the leaves. In stem, collenchyma tissue is located under the epidermis. Figure 6: Cross sections of M. aquatica leaves, (A) peltate glandular trichomes, (B) capitates glandular trichomes. In another study, [20] leaves and stems of M. pulegium were shown to contain simple covering hairs of three cells and two types of glandular trichomes. The first type of glandular 37

5 trichomes is capitate with two cells (stalk and head) and the second type is peltate (gland oval of ten cells covered with large cuticle). In addition, the number of glandular cells diver from one species to another according to stages of growth. Different results were found by Zaks et al [16] in which they mentioned that Lemon mint (M. aquatica var. citrata) contains peltate glandular trichomes that is formed by a stalk cell and eight secretory cells encompassed by an elevated cuticle. The medicinal plant (Mentha aquatica) which was chosen in the present study is one of the most renowned medicinal plants in Algeria. It is known by the ease of hybridization (Mentha genus) and by a high polymorphism in morphology. Its composition of essential oil is diverse according to different regions of the world. Therefore research is carried out in all regions at all times. CONCLUSIONS The composition of essential oil of M. aquatica is diverse according to different regions of the world. However, our analysis of this plant essential oil by GC and GC/MS identified 43 components and the main compounds are the monoterpenes. The anatomical studies carried out on young fresh stems and leaves of mentha aquatica show the presence of two types of trichomes at different developmental stages. The first is the simple hairs (covering trichomes) and the second is the secretive glands. Under light microscope these secretive glands were: big glandular peltate trichomes multicellular oval head and small glandular capitate «unicellular globule head. It would be more beneficial if research is carried out in different region and comparisons are made to gain better understanding of this plant. REFERENCES 1. Gulluce M., Sahin F., Sokmen M., Ozer H., Daferera D., Sokmen A., Polissiou M., Adiguzel A., Ozkan H. Antimicrobial and antioxidant properties of the essential oils and methanol extract from Mentha longifolia L. ssp. longifolia. Food Chemistry 2007; 103: Bhat S., Maheshwari P., Kumar S. and Kumar A. Mentha species: In vitro Regeneration and Genetic Transformation. Molecular Biology Today 2002; 3(1): Allen D. E. et Hatfield G. Medicinal Plants in Folk Tradition An Ethnobotany of Britain and Ireland. Ed. Timber Press Portland.Cambridge Bohloul Abbaszadeh, Sayed Alireza Valadabadi, Hossein Aliabadi Farahani and Hossein Hasanpour Darvishi. Studying of essential oil variations in leaves of Mentha species. African Journal of Plant Science 2009; Vol. 3 (10): Masada Y. Analysis of Essential Oils by Gas Chromatography and Mass Spectrometry,Halsted, Nueva York 1976: p NIST. Mass Spectral Search Program for the NIST/EPA/NIH Mass Spectral Library, vers fiveash data, USA R.P, Adams. Identification of essential oil components by gas chromatography and quadrupole mass spectrometry. Allured Publ. Corp., Carol Stream IL Locquin and Langeron. Manuel de microscopie, Masson, Paris 1978; pp Esmaeili A, Rustaiyan A, Masoudi S, Nadji K. Composition of the essential oils of Mentha aquatica L. and Nepeta meyeri Benth. from Iran. Journal of Essential oil Research: JEOR 2006; 18(3): Agostini F.; Santos A. C. A. d.; Rossato M.; Pansera M. R.; Santos P. L. d.; L. Serafini A.; Molon R.; Moyna P. Essential oil yield and composition of Lamiaceae species growing in southern Brazil. Braz. arch. biol. Technol 2009; vol.52 no.2 : Lamendin H., Toscano G., Rquirand p. Phytothérapie et aromathérapie buccodentaires. EMC-Dentisterie 2004; 1, Znini M., Bouklah M., Majidi L., Kharchouf S., Aouniti A., Bouyanzer A., Hammouti B., Costa J., Al-Deyab S.S. Chemical Composition and Inhibitory Effect of Mentha Spicata Essential Oil on the Corrosion of Steel in Molar Hydrochloric Acid. Int. J. Electrochem. Sci. 2011; 6, Derwich E., Benziane Z., Taouil R., Senhaji O. and Touzani M. Comparative Essential oil Composition of Leaves of Mentha rotundifolia and Mentha pulegium a Traditional Herbal Medicine in Morocco. American-Eurasian Journal of Sustainable Agriculture 2012; 4(1): Mahboubi M., Haghi G. Antimicrobial activity and chemical composition of Mentha pulegium L. essential oil. Journal of Ethnopharmacology 2008; 119, Vian M.A., Fernandez X., Visinoni F., Chemat F. Microwave hydrodiffusion and gravity, a new technique for extraction of essential oils. Journal of Chromatography A 2008; 1190, Zaks A., Davidovich-Rikanati R., Bar E., Inbar M., and Lewinsohn E. Biosynthesis of linalyl acetate and other terpenes in lemon mint (Mentha aquatica var. citrata, Lamiaceae) glandular trichomes. Israel Journal of Plant Sciences 2008; Vol. 56, Chauhan R.S., Kaul M.K., Shahi A.K., Kumar Arun, Ram G., Tawa A. Chemical composition of essential oils in Mentha spicata L. accession [IIIM(J)26] from North-West Himalayan region, India. Industrial crops and products 2009; 29: Tucker Arthur O., Naczi Robert F.C. Mint The Genus Mentha Mentha: An Overview of Its Classification and Relationships. CRC Press Taylor & Francis Group Hatamneia A. A., Khayami M., Mahmudzadeh A., sarghein S. H. and Mahamadaminzade B. Anatomical studies on subfamily Nepetoideae Species (Lamiaceae) in West Azerbijan in Iran. Botany Research Journal 2008; 1 (3): Karray-Bouraoui N., M. Rabhi, M. Neffati, B. Baldan, A. Ranieri, M. Brahim, M. Lachaâl, A. Smaoui. Salt effect on yield and composition of shoot essential oil and trichome morphology and density on leaves of Mentha pulegium. Industrial Crops and Products 2009; 30:

IJPRD, 2011; Vol 3(7): October 2011 (51-55) International Standard Serial Number

IJPRD, 2011; Vol 3(7): October 2011 (51-55) International Standard Serial Number 0974 9446 ----------------------------------------------------------------------------------------------------------------------------------------------------------------